Purification and analysis of biological molecules is very often carried out by forcing these molecules to migrate through a gel. In gel electrophoresis the driving force is a voltage gradient across the gel and the gel matrix comprises natural or synthetic polymers. The synthetic polymers are usually formed by polymerization of double bonds present in monomer and cross-linker molecules.
Electrophoresis is based on the principle that charged molecules or substances will migrate when placed in an electric field. Since proteins and other biopolymers (e.g., DNA, RNA, enzymes and carbohydrates) are charged, they migrate at pH values other than their isoelectric point. The rate of migration depends, among other things, upon the charge density of the protein or biopolymer and the restrictive properties of the electrophoretic matrix. The higher the ratio of charge to mass, the faster the molecule will migrate. The more restrictive the medium, the more slowly an ion will migrate. Electrophoresis has the further advantage of generally requiring only very small (i.e., microgram or less) quantities of material for analysis.
Electrophoresis is generally performed in an aqueous solution or gel across which a voltage is applied. It is the voltage gradient that causes the migration of the species being separated. Gradients typically range from 10 volts/cm to many times higher, the magnitude depending on the nature of the separation being performed.
Many support media for electrophoresis are in current use. The most popular are sheets of paper or cellulose acetate, silica gels, agarose, starch and polyacrylamide. Paper, cellulose acetate, and thin layer silica materials are relatively inert and serve mainly for support and to minimize convection. Separation of proteins using these materials is based largely upon the charge density of the proteins at the pH selected.
On the other hand, starch, agarose and polyacrylamide gel materials not only minimize convection and diffusion but also actively participate in the separation process. These materials provide a porous medium in which the pore size can be controlled to approximate the size of the protein molecules being separated. In this way, molecular sieving occurs and provides separation on the basis of both charge density and molecular size.
The extent of molecular sieving is thought to depend on how closely the gel pore size approximates the size of the migrating particle. The pore size of agarose gels is sufficiently large that molecular sieving of most protein molecules is minimal and separation of proteins is based mainly on charge density. In contrast, polyacrylamide gels can have pores that more closely approximate the size of protein molecules and so contribute to the molecular sieving effect. Polyacrylamide has the further advantage of being a synthetic polymer which can be prepared in highly purified form.
The ability to produce gels having a wide range of polymer concentrations (and, therefore, since the gel network opening decreases with increasing polymer concentration, a wide range of controlled average pore size) as well as to form pore size gradients within the gels by virtue of polymer concentration gradients, are additional advantages of synthetic polymers such as polyacrylamide as electrophoresis gel media. Control over pore size enables mixtures of biological materials to be sieved on the basis of molecular size and enables molecular weight determinations to be performed. These determinations are especially accurate if proteins are treated with a detergent, such as sodium dodecyl sulfate (SDS), which neutralizes the effects of inherent molecular charge so that all SDS treated molecules, regardless of size, have approximately the same charge density values. This technique is referred to as SDS-PAGE (Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis).
Crosslinked polyacrylamide, produced by polymerizing acrylamide containing a few percent of N,N'methylenebisacrylamide (bis), is extensively employed as the matrix for gel electrophoresis. This is due primarily to three properties of the polymer, namely: acceptable mechanical strength, adherence to glass surfaces and wide control of pore size, thereby permitting fractionation of moieties ranging from simple amino acids to complex biological substances having molecular weights in the millions.
The popularity of polyacrylamide-based electrophoresis gels stems not only from the comparatively wide latitude in polymer content and buffer composition attainable with them, but also from the high degree of inertness in the gel with respect to both the voltages applied and the solutes being separated, the ease with which proteins are detected once separated and good reproducibility with carefully prepared gels.
Conventionally, polyacrylamide gel media for use in SDS-PAGE electrophoresis have been prepared in situ by free radical induced polymerization of a monomer such as acrylamide and a crosslinking agent, most commonly N,N'-methylenebisacrylamide, under oxygen-free conditions in the presence of water, a buffer, a polymerization initiator, and a polymerization catalyst. Since such polymerization can be inhibited by the presence of oxygen, polyacrylamide gel media for electrophoresis typically are prepared by a process involving: introducing a previously deoxygenated aqueous solution containing acrylamide, a crosslinking (bis) monomer, a buffer, a free radical polymerization initiator and a polymerization catalyst into a cell formed between two glass plates with a selected clearance (typically about 0.15-5 mm); and sealing the gel-forming solution from oxygen, whereupon the free radical polymerization proceeds so as to prepare the desired gel. Often this is done in situ by the scientist who is to conduct the electrophoresis.
The usual practice is to perform a free radical polymerization with acrylamide and a suitable bis monomer such as N,N'-methylenebisacrylamide (often simply referred to as "bis") in order to obtain a gel. Such gel formation is successfully done only as several precautions are taken namely: (a) very high purity starting materials should be used; (b) the solution of monomers and buffer should be degassed to remove oxygen; (c) a free radical initiator and a catalyst must be quickly mixed in the degassed solution; (d) the solution should be quickly poured between two glass plates or down a glass tube, the lower end of which in either case is sealed to prevent leakage; and (e) the gelation should proceed with (i) oxygen largely excluded and (ii) adequate means for heat dissipation being present so that excess heat does not cause gel nonuniformities.
The cell employed for the preparation of the gel generally has a length of approximately 6 to 60 cm. Accordingly, the introduction of the gel-forming solution into such a long cell requires careful operation to prevent the solution from gelling before it is completely poured (which would prevent the preparation of a uniform polyacrylamide gel medium of the desired length). Thus, the preparation of a polyacrylamide gel medium for electrophoresis having the desired dimensions and consistency requires a great deal of skill and care, as well as keeping the solution free from oxygen.
Precautions are also required in handling the monomers since both acrylamide and bis have been identified as known neurotoxins and suspected carcinogens.
There are several alternatives to the above-described procedure whereby the user makes electrophoresis gels by free radical polymerization and crosslinking in situ. These include (a) the use of preformed gels in cassettes, glass tubing, capillary tubing, and the like; and (b) the use of preformed gels on flexible supports. With either of these alternatives, however, some operating freedom or flexibility with regard to gel size, polymer content in the gel and buffer content is lost. Also, especially with precast gels in cassettes made by free radical polymerization and crosslinking, there generally remain, after completion of the gel formation reaction, some unreacted monomers, initiator by-products and catalyst. The presence of such species poses some toxicological hazards to the user and may interfere with the electrophoretic separation to be performed. Also, such precast gels have been found to have limited shelf lives.
There are, therefore, certain attributes of cross linked polyacrylamide which detracts from its application as an electrophoretic medium. A major concern is that the gel is formed by a polymerization reaction utilizing free radicals, which is exothermic. As is well recognized, free radical reactions depend on a variety of parameters such as concentration of initiators (which themselves tend toward instability), monomer purity, temperature, time, oxygen partial pressure and absence of other inhibitors; managing these factors can require an inordinate amount of care and attention in order to achieve reproducible results. Another at least potential objection to crosslinked polyacrylamide is the possible health hazard from handling of the precursor monomers, acrylamide having been found to be a neurotoxin.
Over the past several years, a great deal of effort has been expended in the investigation and development of electrophoretic gel systems which are free of the problems associated with polyacrylamide.
Accordingly, there is clearly a need in the art for alternative polymer systems for use in separation of biological materials, such as in electrophoresis.